C-Jun N-terminal kinase signaling in the pathogenesis of nonalcoholic fatty liver disease: Multiple roles in multiple steps

Abstract

The c-Jun N-terminal kinase (JNK) is a member of an evolutionarily conserved subfamily of mitogen-activated protein kinases (MAPKs). JNK regulates various cellular responses such as differentiation, proliferation, migration, immune reaction, and cell death in response to a diverse range of extracellular stimuli.1 Gene deletion and pharmacological interventions have revealed that JNK signaling is required for acute hepatocellular injury, liver regeneration, and carcinogenesis.2-7 In addition, JNK plays a central role in obesity and insulin resistance.8 Therefore, JNK has attracted attention as a key regulator in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). AP-1, activator protein-1; ASO, antisense oligonucleotide; ER, endoplasmic reticulum; FFAs, free fatty acids; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α. NAFLD is a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and liver fibrosis, and is commonly associated with clinical features of the metabolic syndrome such as obesity, type II diabetes, and dyslipidemia. A “two-hit” model has been proposed for the development of NALFD.9 The “first hit” is the initial hepatic lipid accumulation, but a “second hit” is required for liver injury and inflammation. JNK has been implicated to play a role in both of these “hits”. First, increased JNK activity can promote the insulin resistance that underlies the development of metabolic syndrome, including hepatic steatosis. Second, hepatic oxidative stress, which is a major candidate for the “second hit”, could cause cellular injury and trigger inflammation through JNK activation. Thus, JNK might play a pivotal role in each step of the pathogenesis of NAFLD. Indeed, recent studies have demonstrated that JNK is activated in the livers of patients with NASH, and that genetic deletion of JNK isoforms attenuates hepatic steatosis in experimental animal models.10-12 Given the potential for JNK inhibition as a possible therapy for insulin resistance/diabetes,13 an in-depth understanding of the role of JNK in the pathogenesis of NAFLD is of critical importance. However, because of the ubiquitous expression of two JNK isoforms (JNK1 and JNK2) and their functional redundancy, compensation, or individuality, it has been difficult to interpret the distinctive roles for JNK isoforms in liver disease. In this issue of HEPATOLOGY, by using the combination of gene knockout and knockdown techniques in mice, Singh and coworkers14 elegantly illustrated the differential contributions of JNK1 and JNK2 in multiple steps of the pathogenesis of NAFLD, and expanded the possibilities of selective inhibition of a JNK isoform as a future therapy for NAFLD. Obesity and insulin resistance are the two major risk factors for NAFLD. In short, systemic insulin resistance, especially at the level of adipocytes, enhances the flux of free fatty acids (FFAs) to the liver. In addition, increased de novo lipogenesis due to hyperinsulinemia induces hepatic steatosis, a “first hit” of NAFLD. This is the differential or selective insulin resistance. JNK plays a central role in both systemic and hepatic insulin resistance through serine phosphorylation of the insulin receptor substrate (IRS)-1 and IRS-2, leading to down-modulation of tyrosine phosphorylation of these molecules, which is required for normal insulin signaling.8, 15 In fact, JNK activation induced by various stimuli such as obesity-induced inflammation, FFAs, oxidative stress, or endoplasmic reticulum (ER) stress, mediates insulin resistance and subsequent hepatic steatosis. Singh and coworkers investigated the distinct roles for JNK1 and JNK2 in developing or established insulin resistance and hepatic steatosis by using genetic knockout or antisense oligonucleotide (ASO) knockdown techniques in mice fed a high-fat diet, respectively. In addition to reproducing the previously reported decreased incidence of insulin resistance in jnk1−/− but not in jnk2−/− mice, they found that ASO knockdown of either JNK1 or JNK2 is able to reverse the established insulin resistance, mimicking a gene targeting therapy. Given that ASOs are preferentially delivered to the liver, JNK ablation in the liver may improve not only hepatic but also systemic insulin sensitivity. Furthermore, they demonstrated that ablation of JNK1 but not JNK2 improved hepatic steatosis. The differential effect of JNK2 knockdown, which is effective on insulin resistance but not on hepatic steatosis, highlights that hepatic steatosis is more than a mere consequence of insulin resistance. JNK1 might have a direct effect on hepatic lipogenesis that is independent of insulin resistance. A previous article supports these results, in that ASO treatment against JNK1 reduces hepatocyte lipid production in vitro and hepatic steatosis in vivo (Fig. 1).16 Multiple roles of JNK isoforms in the pathogenesis of NAFLD. In the setting of systemic insulin resistance, hyperinsulinemia and hyperlipidemia contribute to the development of hepatic steatosis by de novo lipogenesis and increased FFA flux to the liver, respectively. Steatosis-induced oxidative stress, ER stress, and lipid peroxidation activate JNK. Both JNK1 and JNK2 cause hepatic insulin resistance by serine phosphorylation of IRS-1 and IRS-2, whereas only JNK1 induces steatosis. Oxidative stress and proinflammatory cytokines mediate hepatocyte injury through JNK activation. JNK1 and JNK2 indirectly activate caspase-8. JNK2 also plays a cytoprotective function by inhibiting the Bim-dependent mitochondrial pathway of apoptosis. JNK promotes the development of inflammation through AP-1–dependent transcription. JNK1 in inflammatory cells promotes inflammation. According to the “two-hit” hypothesis for NAFLD progression, a “second-hit' is required for the actual hepatocellular injury. A major candidate for the “second hit” is hepatic oxidative stress, which is induced by excess FFAs, associated mitochondrial dysfunction, or lipid peroxidation. Although early and transient activation of JNK promotes cell survival, sustained activation induced by reactive oxygen species via inactivation of the mitogen-activated protein kinase phosphatases contributes to cell death.17 Many factors other than oxidative stress, such as FFAs, ER stress, and inflammatory cytokines including tumor necrosis factor-α directly activate JNK and accelerate cellular injury in NAFLD. The distinct roles for JNK1 and JNK2 in hepatocyte death are still controversial. JNK1 phosphorylates and activates Itch, which induces ubiquitination/degradation of c-FLIP (cellular FLICE-inhibitory protein) and subsequent caspase-8–dependent apoptosis, whereas JNK2 may activate caspase-8 more directly.3, 4 In a cell culture model of NAFLD, FFA-induced JNK activation in primary hepatocytes activates proapoptotic Bcl-2 (B-cell lymphoma-2) family members Bim and Bax and induces cytotoxicity at the mitochondrion level.6 On the other hand, Singh and coworkers found that JNK2 ablation in mice fed a high-fat diet augments hepatocellular injury independent of the level of hepatic steatosis. They propose that JNK2 plays a cytoprotective function by inhibiting Bim-dependent apoptosis through phosphorylation and degradation of Bim. Together with a cytoprotective function of JNK1 through phosphorylation and stabilization of antiapoptotic Bcl-2 family member Mcl-1 (Y.K. and D.A.B., unpublished data), each JNK isoform exhibits both proapoptotic and antiapoptotic effect in the hepatocyte depending on stimulation, time-course, or other circumstances of the cell (Fig. 1). Another important aspect of NAFLD is chronic hepatic inflammation, which is a key event for the progression of the disease. In the steatotic liver, many factors such as oxidative stress, ER stress, and lipid peroxidation can directly activate the inhibitor of nuclear factor-κB (NF-κB) kinase or JNK to activate transcription of proinflammatory cytokines through NF-κB or activator protein-1 (AP-1), respectively. In addition to obesity-induced inflammation derived from adipose tissue, increased serum FFAs and increased portal vein lipopolysaccharide concentrations from small intestinal bacterial overgrowth may activate NF-κB and JNK through Toll-like receptor signaling. This hepatic and systemic inflammation enhances insulin resistance, hepatic steatosis, and liver injury, leading to the progression of steatohepatitis to liver fibrosis. Although JNK1 deletion diminishes hepatic inflammation in animal models, it is unclear whether this is a direct effect of JNK1 deletion on inflammatory signaling or secondary effect through less obesity and hepatic steatosis.11, 12 Solinas et al. demonstrated that selective JNK1 deletion in hematopoietic cells prevents systemic and hepatic inflammation and subsequent development of insulin resistance induced by a high-fat diet without affecting the level of obesity or hepatic steatosis.18 Similarly, JNK1 deletion in hematopoietic cells prevents hepatic steatosis-induced liver inflammation and fibrosis despite a similar level of steatosis, induced by choline-deficient L-amino acid–defined diet (Y.K. and D.A.B., unpublished data). Taken together, JNK1 contributes to the development of inflammation in NAFLD not only through insulin target cells such as adipocytes and hepatocytes, but also through activating inflammatory signaling in hematopoietic cells (most likely Kupffer cells) (Fig. 1). Thus, both JNK isoforms play critical roles in each step of insulin resistance, hepatic steatosis, liver injury, and inflammation in the pathogenesis of NAFLD. The article by Singh et al. provides new insights into the functions of JNK isoforms in multiple steps of NAFLD pathogenesis by utilizing an ASO knockdown system in addition to knockout mice. In particular, the finding that JNK1 ablation is effective in treating established insulin resistance and hepatic steatosis, whereas JNK2 ablation exacerbates liver injury, is extremely important in exploring future therapy. This raises an alarm over indiscriminate JNK inhibition and underscores that the selective inhibition of the JNK1 isoform could be a very effective treatment of NAFLD. At the same time, new questions arise from the research of Singh and coworkers. How do JNK isoforms regulate lipogenesis in the liver? Do the isoforms compensate each other when one of them is knocked down? Is cell lineage–specific JNK1 ablation required for NAFLD therapy? Further development of time-specific and cell lineage–specific knockdown systems will provide answers for these questions and further extend the possibility of JNK isoform targeting therapy. We thank Dr. Jerrold M. Olefsky, University of California, San Diego, for helpful discussions.